The present invention relates to isolated nucleotide sequences useful for the production of plants with a modified embryo and/or endosperm development, to vectors containing the nucleotide sequences, to proteins encoded by the nucleotide sequences, to methods for obtaining the nucleotide sequences, to methods for isolating embryo sac-specific genes or proteins from a plant and to methods for producing agronomically interesting plants exhibiting female sterility or allowing apomictic propagation.
Diploid sporophytic and haploid gametophytic generations, alternate in the life cycle of higher and lower plant species. In contrast to lower plant species such as mosses or green algae in which the haploid gametophyte is the dominant generation, the gametophyte in higher plant species is dramatically reduced (Reiser and Fischer, 1993; Drews et al., 1998). Both male (pollen) and female (embryo sac) gametophytes have developed from spores, the haploid products of meiosis from spores (micro- and megaspores). In angiosperms, male gametophytes (pollen) are simple two to three-celled organisms consisting of one vegetative and one or two sperm cells, which are species-specific (Bedinger, 1992; McCormick, 1993). Three of the four megaspores in most angiosperms degenerate and the surviving one forms the female gametophyte after three mitotic divisions (Reiser and Fischer, 1993; Russel, 1993). The predominant female gametophyte, the Polygonium type, which occurs in about 70% of the angiosperm species (Webb and Gunning, 1990; Reiser and Fischer, 1993), is deeply embedded in sporophytic tissue and consists of only seven cells: the egg cell, two synergids, a central cell and three antipodals. In maize and several other species, the antipodal cells continue to proliferate until a group of about 20 to 40 cells is formed (Kiesselbach, 1949).
The main function of the gametophytes is to supply the gametes: male and female gametes fuse during fertilisation, combine their different genomes, and thus form a new sporohytic generation. Thus, sexual reproduction in angiosperms is initiated when pollen grains start to germinate on the female flower organ, the stigma (Cheung, 1996). The female gametophyte might then function in (i) directing the pollen tube to the ovule (Hülskamp et al., 1995; Ray et al., 1997), (ii) directing one sperm cell to the egg cell and the other to the central cell (Russel, 1992), (iii) generating a barrier to polyspermy (Faure et al., 1994; Kranz et al., 1995), (iv) preventing autonomous embryo (parthenogenesis) and endosperm development (Grossniklaus et al., 1998; Luo et al., 1999; Ohad et al., 1999) and finally (v) accumulating stores of maternal mRNAS to facilitate the rapid initiation of embryo and endosperm development after fertilisation (Dresselhaus et al., 1999b).
Morphological and structural studies of female gametophyte development as well as fertilisation and early embryo/endosperm development have been employed with many plant species (e.g. with maize: Kiesselbach, 1949; Diboll 1968; Huang and Sheridan, 1994 and Arabidopsis: Webb and Gunning, 1990, 1991; Muriga et al., 1993). In contrast, “The identities and specific functions of the haploid-expressed genes required by the female gametophyte are almost completely unknown” (Drews et al., 1998). This reflects the technical difficulty of identifying mutants and of gaining access to certain developmental stages for molecular analyses.
Many mutants have been described that affect female gametophyte development and function, especially in maize and Arabidopsis, suggesting that a large number of loci is essential for embryo sac development (Vollbrecht and Hake, 1995; Drews et al. 1998; Grossniklaus and Schneitz, 1998. A few maternal genes functioning in the embryo sac as repressors of autonomous embryo (pathenogenesis) and/or endosperm development have been recently cloned in Arabidopsis. Mea/fis1 (medea/fertilisation independent seed 1) is a gametophyte maternal effect gene probably involved in regulating cell proliferation in endosperm and partially in the embryo as well (Grossniklaus et al., 1998; Luo et al., 1999). Fis3 shows a similar phenotype and encodes a putative zinc-finger transcription factor (Luo et al., 1999): Autonomous endosperm development was observed in the fie (fertilisation independent endosperm/fis3 mutant. Mea/fis1 and fie/fis3 display homology to polycomb proteins (Grossniklaus et al. 1998; Ohad et al., 1999), proteins which are involved in long-term repression of homeotic genes in Drosophila and mammalian embryo development (Pirrotta, 1998).
At a low frequency, auxin (2, 4 D) treated sexual eggs from maize can be triggered to initiate embryo development (Kranz et al., 1995), and some egg cells initiate parthenogenetic development spontaneously. In wheat, lines have been described producing up to 90% parthenogenetic haploids (Matzk et al., 1995). The molecular mechanisms underlying these processes are completely unknown. One protein (α-tubulin) was identified whose expression is associated with the initiation of parthenogenesis in wheat (Matzk et al., 1997). De novo transcription from the zygotic genome occurs relatively soon after fertilisation in maize (Sauter et al., 1998; Dresselhaus et al., 1999a), indicating that the store of maternal mRNA and the maternal control of embryo development is not as relevant as it is in animal species, for example Drosophila, Xenopus or Zebrafish (Orr-Weaver 1994; Newport and Kirschner 1982; Zamir et al. 1997).
An important biological process linked to flower and seed development is apomixis (asexual reproduction through seeds: Koltunow et al., 1995; Vielle-Calzada et al., 1996). Due to the enormous economical potential of apomixis once controllable in sexual crops, its application was named after the ‘Green Revolution’ as the ‘Asexual Revolution’ (Vielle-Calzada et al., 1996). Up to now all approaches to isolate the ‘apomixis genes’ from apomictic species failed. Genes involved in autonomous endosperm development once inactivated were recently isolated from Arabidopsis (see Ohad et al., 1999; Luo et al., 1999). Autonomous embryo development (via parthenogenesis), a further component of apomixis will be necessary to engineer the apomixis trait in sexual crops. E.g. in wheat, lines have been described producing up to 90% parthenogenetic haploids (Matzk et al., 1995). Almost no molecular data concerning parthenogenesis is available for higher plants: one protein (α-tubulin) was identified from the above described wheat lines whose expression is associated with the initiation of parthenogenesis (Matzk et al., 1997). Nevertheless, such a ‘house keeping gene’ will not be a valuable tool for genetic engineering of the induction of parthenogenesis. Regulatory genes are needed.
Thus, from an agronomical point of view it is highly desirable to provide plants; in particular agronomically important plants, which allow improved hybrid breeding, apomictic propagation and/or plants having seedless fruits, as well as providing female sterile plants.
Thus, it is considered particularly important to develop and provide means and methods that allow the production of plants exhibiting a modified embryo and endosperm development, in particular plants exhibiting a modified female gametophyte development. Such plants may prove particularly useful in commercial breeding programmes.